Cyclic AMP Sensitive Signalling by the CD28 Marker Requires Concomitant Stimulation by the T-Cell Antigen Receptor (TCR/CD3) Complex

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RIα₂-Cβ₂) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anli-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP)     

Inhibition of programmed cell death by cyclosporin A; preferential blocking of cell death induced by signals via TCR/CD3 complex and its mode of action.

Cyclosporin A (CsA) is reported to inhibit programmed cell death. We confirmed this by using T-cell hybridomas which are inducible to programmed cell death by activation with immobilized anti-CD3 antibody or with anti-Thy 1.2 antibody. Cell death and DNA fragmentation, characteristic features of programmed cell death, were almost completely blocked by CsA or FK506. To investigate whether CsA inhibits only the cell death through the signals via the TCR/CD3 complex or all of the programmed cell death induced by various reagents, we further established CD4+8+ thymic lymphomas which result in programmed cell death after activation with calcium ionophore, dexamethasone, cyclic AMP or anti-CD3 antibody. It was revealed that CsA could block only the cell death mediated by the TCR/CD3 complex. For the clarification of the site of action of CsA, Ca2+ influx and endocytosis of receptors after stimulation with anti-CD3 antibody were monitored in the presence of CsA, and no significant effects of CsA were observed. Furthermore, prevention of cell death was examined by adding CsA at various periods of time after initiation of culture. CsA was found to exert its effect even when added after 4 h of cultivation, and the kinetic pattern of suppression was similar to that of the suppressive effect on IL-2 production. These observations indicate that in the events of programmed cell death, the major site of action of CsA will not be the inhibition of the immediate membrane events after activation of the TCR/CD3 complex but rather the interference in the function of molecules that transmit signals between membrane events and the activation of genes in the nucleus.     

Persistent CD3-Crosslinking Down-Regulates Interleukin-2 Responsiveness in Interleukin-2-Competent Cloned T Cells: the Possible Involvement of Protein Kinase C

To investigate the regulation of interleukin-2 (IL-2) responsiveness of T cells, a human CD4⁺ T-cell clone with constitutive expression of IL-2 receptors was stimulated with recombinant IL-2 (rIL-2) in the presence or absence of immobilized anti-CD3 monoclonal antibodies (αCD3_imm MoAb). Incubation of T cells with αCD3_imm MoAb decreased IL-2-induced proliferation which could not be ascribed to the modulation of IL-2 receptor expression nor to cell death. Phorbol-myristate-acetate (PMA), an activator of protein kinase C (PKC), also induced down-regulation of IL-2 responsiveness. The αCD3_sol MoAb, inducing Ca²⁺-mobilization without activating PKC, did not inhibit IL-2 responsiveness whereas cyclosporine A (CsA), a drug that inhibits the Ca²⁺-dependent activation pathway, did not prevent the induction of IL-2 hyporesponsiveness induced by αCD3_imm MoAb. It is concluded that modulation of IL-2 responsiveness of T cells via the T-cell receptor/CD3 complex (TCR/CD3) may be mediated by a PKC-activating signal.     

Immobilized anti-CD5 together with prolonged activation of protein kinase C induce interleukin 2-dependent T cell growth: evidence for signal transduction through CD5.

Monoclonal antibodies (mAb) identifying the CD5 antigen were used to stimulate human peripheral blood T lymphocytes. Three out of three anti-CD5 mAb, 10.2, OKT1 and anti-Leu-1 induced vigorous proliferation of purified T cells in the presence of 1.6 nM phorbol 12-myristate 13-acetate (PMA). Immobilization of anti-CD5 mAb on a solid support was necessary for the induction of a proliferative response. Neither 1.6 nM PMA, nor immobilized anti-CD5 mAb were mitogenic as a sole stimulus. mAb identifying CD4, CD7, CD11a, CD18, and major histocompatibility complex class I molecules were not comitogenic with PMA. Anti-CD5/PMA-induced cell proliferation proceeded by an interleukin 2 (IL 2)-dependent mechanism, as was demonstrated by the cell surface expression of the p55 chain of the IL 2 receptor (IL 2R), the production of IL 2 and the inhibition of the proliferative response by anti-IL 2R mAb anti-Tac. There was no strict requirement for detectable numbers of monocytes, although cell proliferation could be enhanced by the monocyte-derived cytokines IL 1 and IL 6. Phorbol 12,13-dibutyrate and mezerein could substitute for PMA in this activation pathway, but synthetic diacylglycerols and phorbol esters that do not activate protein kinase C (PKC) could not, indicating a need for prolonged activation of PKC. T cells activated by anti-CD5/PMA are sensitive to inhibition by cyclosporin A (CsA) and by prostaglandin E2 (PGE2). This contrasts with anti-CD28/PMA-induced T cell proliferation, which is resistant to CsA and PGE2. Cell surface expression of CD5 was strongly up-regulated by PMA, whereas CD3 expression was down-regulated. We conclude that T cell activation can be triggered by engagement of CD5 by immobilized anti-CD5 mAb, combined with prolonged activation of PKC. These data support a role for CD5 as an independent signal transducing molecule.     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

Regulation of T-cell differentiation by CD2 and CD28 accessory molecules.

It was analysed to what extent the functional T-cell responses that result from T-cell receptor (TcR)/CD3 triggering differ from responses that are induced after simultaneous ligation of CD2 and CD28 accessory molecules. To allow a quantitative comparison of these activation pathways, purified lymphocytes were stimulated with either graded densities of immobilized anti-CD3 monoclonal antibodies (mAb) or with increasing amounts of anti-CD28 mAb in the presence of a constant concentration of anti-CD2 mAb. Both activation systems were sensitive to the regulatory properties of CD11a/CD18 molecules. T-cell stimulation via CD2/CD28 molecules induced a more potent release of interleukin-2 (IL-2) and more pronounced T helper (Th) cell responses than T-cell stimulation via the TcR/CD3 complex, whereas CD25 expression was more readily initiated after T-cell activation via the TcR/CD3 complex. Optimal Th cell differentiation was detected under conditions of suboptimal receptor occupancy whereas, in contrast, optimal cytolytic T lymphocyte (CTL) differentiation required optimal TcR/CD3 or CD2/CD28 engagement. The findings indicate that T-cell differentiation can be influenced in a qualitative manner by the strength of the activation signal provided, and suggest that antigen-specific T-cell responses might be regulated in a quantitative manner through CD2 and CD28 accessory molecules.     

Cyclosporin A increases IFN-γ production by T cells when co-stimulated through CD28

Despite its calcineurin-inhibiting properties, cyclosporin A (CsA) can not inhibit IL-2 production when T cells are co-stimulated by CD80/CD86 on the antigen-presenting cells. We studied the in vitro effect of CsA on IFN-γ production. Anti-CD3 monoclonal antibody (mAb) was used as the primary stimulus for activation of purified human T cells. A stimulating anti-CD28 mAb, or CD80 or CD86 on stably transfected P815 cells, provided the co-stimulatory signal. IL-2 production was hardly affected by CsA under these stimulating conditions, while IFN-γ (at the protein and mRNA level) was markedly stimulated by CsA. The use of anti-CD3 or phorbol 12-myristate 13-acetate with ionomycin as the primary stimulus, together with co-stimulation through either CD28 or CD2 using transfectants with the appropriate ligands, allowed us to demonstrate that the resistance of IFN-γ production to inhibition by CsA required both CD3 and CD28 triggering. Inhibition of IL-10 production, and to a lesser degree of IL-4 production, by CD4⁺ cells was responsible for the enhancement of IFN-γ production in the presence of CsA. In conclusion, IFN-γ production by CD28-co-stimulated CD4⁺ T cells is resistant to inhibition by CsA and can even be facilitated by CsA as a result of removing a negative regulatory signal which is mainly IL-10 mediated. This finding might have implications for immunosuppressive strategies based upon the use of CsA.     

Interleukin-12 can replace CD28-dependent T-cell costimulation during nonspecific cytotoxic T lymphocyte induction by anti-CD3 antibody

Cytotoxic T lymphocyte (CTL) development is regulated closely by an intricate series of signals provided by the T-cell receptor/CD3 complex, cytokines, and costimulatory ligand/receptor systems. In this study, we have explored the role of interleukin (IL)-12 and CD28 in mouse CTL development. Activation of T cells with anti-CD3 monoclonal antibody (mAb) in the presence of anti-CD86 mAb, which prevents CD28-CD86 interaction, led to decreased production of type 1 (IL-2, interferon-gamma) and type 2 (IL-4, IL-6, IL-10) cytokines, as well as diminished expression of granzyme B (Gzm B) and reduced cytotoxic effector function. Cytolytic activity in T-cell cultures that were activated in the presence of anti-CD86-blocking mAb alone or in combination with anti-CD80 mAb could be restored by the addition of exogenous IL-12 at initiation of culture. The ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development was found to be dependent on the presence of IL-2 rather than interferon-gamma. IL-2 is required for IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb. Moreover, IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb is enhanced by IL-12. We, therefore, conclude that the ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development is a result of the interaction of IL-12 with IL-12Rbeta2 induced by low levels of IL-2 synthesized by T cells activated in a CD28-independent manner.     

Activation of peripheral CD8+ T lymphocytes via CD28 plus CD2: Evidence for IL-2 gene transcription mediated by CD28 activation

It is well established that peripheral CD8+ and CD4+ T cells display different requirements for in vitro activation by mitogenic mAb. Most CD4+ T cells can be activated by anti-CD3 or mitogenic combinations of anti-CD2. In contrast, CD8+ T cells display minimal responses to CD3 activation, and no proliferation is observed via CD2 activation. Purified peripheral blood CD8+ T cells, stringently depleted of APC, have been studied for their capacity to respond to mAb directed against CD3, CD2 and CD28, used alone or in combination. It is demonstrated that proliferation can be induced by co-stimulation of CD2 and CD28. This does not require autologous APC. CD8+ T cells can also be activated by the combination of anti-CD3 plus anti-CD28 in the presence of APC, but only minimal cell proliferation is obtained in the absence of APC. The response via CD2 plus CD28 is IL-2-dependent, as demonstrated by the ability of mAb against the IL-2 receptor to block proliferation, and is almost completely inhibited by cyclosporine A (CsA). These results suggest that the signal generated by stimulation of CD28 in combination with CD2 differs from that seen with CD28 activation combined with either PMA or CD3. Induction of IL-2 gene activation in CD8+, CD28+ peripheral T cells may therefore require additional "second signals", which are not necessary for activation of CD4+ cells. One such signal might be the interaction between CD28 and its natural ligand.     

Triggering CD 28 molecules synergize with CD 2 (T 11.1 and T 11.2)-mediated T cell activation

Pairs of monoclonal antibodies (mAb) defining epitopes T 11.1 and T 11.2 on the CD 2 molecule are mitogenic for purified human T cells in the presence of a submitogenic dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Anti-CD 28 mAb can substitute for the action of TPA in the anti-CD 2-induced proliferative response of resting T cells, whereas each signal alone is unable to mediate this effect. Co-stimulation by anti-CD 2 plus anti-CD 28 mAb is monocyte independent and besides resting T cells also induces strong proliferation of thymocytes and pre-activated T cells. Modulation of the CD 3-T cell receptor complex does not inhibit the co-stimulatory effects of anti-CD 2 plus anti-CD 28 mAb. The effect is largely dependent on endogenously produced interleukin 2, since the response is strongly inhibited in the presence of mAb against the 55-kDa interleukin 2 receptor chain.     

Signaling via CD28 costimulates lymphokine production,but does not reverse unresponsiveness to interleukin-2 in anti-CD3 triggered Th1 cells

Previously, it has been described that the ability of murine T_h1 cells to proliferate in response to exogenous interleukin (IL)-2 is blocked when these cells are exposed to immobilized anti-CD3 antibodies. In the present study we examined whether simultaneous triggering of the T cell antigen CD28 can prevent the induction of unresponsiveness to IL-2 in T_h1 cells. We report that costimulation of T_h1 cells with anti-CD28 monoclonal antibodies (mAb) did not overcome unresponsiveness to IL-2 induced by various amounts of immobilized anti-CD3 antibodies. However, stimulation with anti-CD28 mAb strongly augmented IL-2 and interferon-γ production in anti-CD3-exposed T_h1 cells. Thus, despite the fact that anti-CD28 mAb is a potent costimulus for lymphokine production, signaling through CD28 does not seem to be sufficient to trigger proliferation in T_h1 cells activated via the T cell receptor. These data suggest the existence of at least three signals to trigger T_h1 cell activation. The first is mediated by ligation of the T cell receptor. One cosignal, delivered by the CD28 molecule, leads to IL-2 production. A third, still undefined, signal is required for proliferation in response to IL-2.     

Non-responsiveness of antigen-experienced CD4 T cells reflects more stringent co-stimulatory requirements.

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.     

Ligation of the CD5 or CD28 molecules on resting human T cells induces expression of the early activation antigen CD69 by a calcium- and tyrosine kinase-dependent mechanism.

The CD5 and CD28 molecules on T lymphocytes can each exert an accessory role in T-cell activation. Ligands for CD5 and CD28 have been identified as CD72 and B7/BB1 respectively. The function of, and the signal transduction pathways coupled to CD28 have been the subject of extensive studies. In contrast, it is still debated whether CD5 functions as a receptor which directly transduces an independent signal to the T cell. In this paper, it is reported that culture of purified T cells in the presence of either immobilized anti-CD5 monoclonal antibody (mAb) (OKT1, Leu-1 or 10.2) or cross-linked anti-CD28 (9.3) mAb (but not of anti-LFA-1 alpha, anti-LFA-1 beta, or anti-CD7) induces expression of CD69, an early activation marker, in the absence of other activating stimuli. CD69 expression was consistently detectable after 3-24 hr on 20-50% of T cells, within both the CD4 and CD8 subsets. CD45RO- CD45RA+ naive T cells were more responsive than CD45RO+ CD45RA- memory T cells. In the presence of recombinant (r) interleukin-2 (IL-2), anti-CD5- or anti-CD28- induced CD69 expression was further up-regulated, more sustained and, as previously shown, succeeded by IL-2 responsiveness. Simultaneous cross-linking of both CD5 and CD28 enhanced CD69 expression above the levels obtained with optimal amounts of both ligands separately. In the presence of a submitogenic dose of the protein kinase C (PKC) activating agent phorbol 12-myristate 13-acetate (PMA), co-stimulation with anti-CD5 or anti-CD28 increased CD69 expression above that induced by PMA alone. Cross-linking of CD5 or CD28 induces an early rise of cytoplasmic free calcium concentration ([Ca2+)]i) and both this rise and CD69 expression were inhibited by chelation of extracellular Ca2+ with ethyleneglycol-bis-(2-aminoethyl)-tetraacetate (EGTA). Pretreatment of the cells with the tyrosine kinase inhibitor herbimycin A also blocked CD69 expression. The data thus antigen-independent fashion. Moreover it is demonstrated that influx of Ca2+ and tyrosine kinase activity are involved in the signal transduction pathways of both receptors.     

Blocking CD40 - CD154 and CD80/CD86 - CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production.

Although CD28 triggering provides an important co-stimulatory signal to T cells, blocking the CD80/CD86 - CD28 interaction with CTLA-4lg fusion protein is not sufficient for tolerance induction in vivo or in vitro. According to more recent data, interruption of the CD40 - CD154 interaction might complement the effect of CTLA-4lg and induce graft acceptance. We studied the effects of a blocking anti-CD40 monoclonal antibody (mAb) and/or blocking anti-CD80/anti-CD86 mAb in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with allogeneic PBMC. T cells activated by alloantigens in the presence of anti-CD80, anti-CD86 and anti-CD40 entered a state of alloantigen-specific non-responsiveness as evidenced upon restimulation by lack of proliferation, cytotoxic activity, and IL-2, IL-5 and IL-13 production. IFN-gamma production during restimulation was less than in the control cultures, while the production of IL-10 was enhanced. Addition of recombinant IL-2 during the restimulation rescued alloantigen-specific activity. We conclude that the simultaneous blocking of the CD40 - CD154 and CD80/CD86 - CD28 interaction during allogeneic T cell activation induces T cell anergy. Since anergic cells induced by this treatment still produce high levels of IL-10, the latter could contribute to modulation of antigen-presenting cell activity and to bystander suppression of residual reactive T cells.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

环孢素A对小鼠脾细胞IFN-γ产生的影响

目的研究在不同刺激条件下,环孢素A(CsA)对小鼠γ-干扰素(IFN-γ)产生的影响。方法小鼠脾淋巴细胞分别使用抗CD3单克隆抗体(mAb)、抗CD3mAb 抗CD28mAb及抗CD3mAb rIL-12刺激后,用ELISA法检测CsA对小鼠脾细胞IFN-γ产生的影响。在单个细胞水平上,用流式细胞仪检测CsA对脾细胞表面CD25和CD69表达及IFN-γ产生的影响。结果CsA对不同条件下诱导的小鼠脾细胞IFN-γ产生,均表现为剂量依赖性的抑制作用,其抑制机制与细胞的活化有关。结论CsA可以剂量依赖的方式抑制小鼠脾细胞表达CD25、CD69及产生IFN-γ。     

Induction of abortive and productive proliferation in resting human T lymphocytes via CD3 and CD28

How the T-cell receptor (TCR)/CD3 complex mediates positive as well as negative signals for T-cell regulation is not fully understood. We have previously described the induction of anergy in resting human T lymphocytes after mitogenic, high-dose CD3 triggering with monoclonal antibodies. Here we report the concomitantly occurring cell death to be largely caused by apoptosis, which mainly affects the CD4+ T-helper population. Because cell death becomes detectable after initial cell proliferation, it appears that a high-dose CD3 stimulus constitutes a negative signal for resting T cells leading to an 'abortive proliferation' that is followed by anergy and/or apoptosis of the cells. In contrast, if initial proliferation is induced by a low dose of anti-CD3 in the presence of an accessory signal via the CD28 receptor, anergy and cell death are markedly reduced and 'productive proliferation' may occur. Productive proliferation is characterized by an increased secretion of various cytokines measured (interleukin (IL)-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma)). A low-dose submitogenic CD3 stimulus induced neither anergy nor cell death, supporting the view that negative CD3 signalling requires proliferation of resting cells.     

IL-10 directly acts on T cells by specifically altering the CD28 co-stimulation pathway

IL-10 induces T cell anergy in numerous mouse models and specific immunotherapy of allergy in humans. Here, we demonstrate that IL-10 directly acts on T cells which are stimulated via CD28 by efficiently blocking proliferation and cytokine production. T cells tolerized by IL-10 showed high viability and the unresponsive state was reversed by anti-CD3 monoclonal antibody (mAb) stimulation and IL-2, but not by anti-CD28 mAb stimulation. Signal transduction via CD28 requires CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3-kinase. IL-10 inhibited tyrosine phosphorylation of CD28; thus, the phosphatidylinositol 3-kinase binding to CD28 was blocked. Consequently, IL-10 inhibited the antigen-induced secretion of both Th1 and Th2 cytokines, including IL-2, IFN-gamma, IL-4, IL-5 and IL-13. Furthermore, neutralization of endogenously produced IL-10 significantly increased T cell proliferation and both Th1 and Th2 cytokine production in vitro. Using superantigen stimuli, T cell suppression by IL-10 was merely induced at low doses when co-stimulation by CD28 was essential. Together, these data demonstrate that IL-10 directly acts on the CD28 signaling pathway and this represents an important T cell suppression mechanism leading to anergy.     

协同刺激分子4-1BB和CD28对人外周血不同T细胞亚群的激活作用

目的 :探讨 4 1BB/ 4 1BBL协同刺激信号在CD4 和CD8 T细胞活化、增殖中的作用 ,并与CD2 8/B7信号作比较。方法 :用抗CD3单抗 (mAb)刺激人外周血单个核细胞 (PBMC)。用阻断型抗 4 1BBLmAb和抗CD80mAb ,分别阻断 4 1BB/ 4 1BBL和CD2 8/B7 1协同刺激信号。利用流式细胞术 (FCM)检测CD4 T细胞、CD8 T细胞的增殖率、CD8/CD4T细胞的比值变化和细胞分泌IFN γ的情况。结果 :用抗 4 1BBLmAb和抗CD80mAb阻断相应的协同刺激途径后 ,CD4 和CD8 T细胞的增殖和细胞分泌IFN γ的水平均明显下降。培养 8d,抗CD3mAb单独刺激组CD8/CD4T细胞的比值为 1.98± 0 .0 6 ;抗 4 1BBLmAb阻断组CD8/CD4T细胞的比值下降为 0 .96±0 .0 3;而在抗CD80mAb阻断组 ,其比值上升为 2 .6 9± 0 .16。结论 :4 1BB分子可在CD4 T细胞和CD8 T细胞的活化、增殖中提供协同刺激信号。 4 1BB分子所介导的协同刺激信号 ,在CD8 T细胞活化及增殖中发挥了更为重要的作用 ;而CD2 8分子所介导的协同刺激信号则更有利于CD4 T细胞的活化